1. Sources of Heavy Metal Pollution in the Environment

Naturally occurring heavy metals are present in forms that are not readily available for uptake by plants. They are typically present in insoluble forms, like in mineral structures, or in precipitated or complex forms that are not readily available for plant uptake. Naturally occurring heavy metals have a great adsorption capacity in soil and are thus not readily available for living organisms. The bonding energy between naturally occurring heavy metals and soil is very high compared to that with anthropogenic sources. Examples of natural processes that bring about the occurrence of heavy metals in the environment are comets, erosion, volcanic eruptions, and the weathering of minerals. Heavy metals from anthropogenic sources typically have a high bioavailability due to their soluble and mobile reactive forms. These anthropogenic sources include alloy production, atmospheric deposition, battery production, biosolids, coating, explosive manufacturing, improper stacking of industrial solid waste, leather tanning, mining, pesticides, phosphate fertilizer, photographic materials, printing pigments, sewage irrigation, smelting, steel and electroplating industries, textiles, and dyes and wood preservation ^[1][2][1,2]. Sources of heavy metals, concentrations in soil, soil properties, the degree and extent of uptake by plants, and the extent of absorption by animals, are the factors that influence the accumulation of metal ions in the food web^[3] [3]. According to D’amore, et al.^[4] [4], the geochemical cycle of heavy metals results in the buildup of heavy metals in the environment, which could cause risk to all life forms when they are above permitted levels. The routes of entry into the environment usually include the weathering of parent materials, the alteration of the geochemical cycle by man, soil ingestion (which is the primary exposure route to humans of soil-borne metals), the transfer from mines to other locations, and the discharge of high concentrations of metal waste by industries.

Mining has negatively affected the environment, causing destruction and an alteration of the ecosystem, including a loss of biodiversity and an accumulation of pollutants in the environment. Mining and ore processing are major sources of heavy metal pollution in the soil, and the recovery of ecosystems from mining activities could take several decades. These activities produced large quantities of stockpiles and dumps, which are frequently abandoned without treatment. Abandoned mines contaminate water bodies through chemical run-off and particulates that accumulate in water sources^[5] [5], hence, creating a need to treat wastewaters contaminated with heavy metals, before discharge into the environment occurs.

2. Toxicity of Heavy Metals to Life Forms

Although some heavy metals play important roles in the physiological, biochemical, and metabolic processes of living organisms, functioning as co-factors for some enzymes, micronutrients, regulators of osmotic pressure, and stabilization of molecules, the majority of them have no known biological function in living organisms and are toxic when generated in excess^[6] [6]. The toxicity of metals is the ability of a metal to cause undesirable effects on organisms. This depends on the heavy metal bioavailability and the absorbed dose^[7] [7]. The threat posed by heavy metals to the health of living organisms is worsened by their continuously persistent nature in the environment. Toxicity increases when the medium becomes acidic and nutrient-deficient, and when the soil structure is poor, especially in mining environments^[8] [8].

Metal toxicity is also shown in their ability to disrupt enzyme structures and functions by binding with thiol and protein groups, or by replacing co-factors in prosthetic groups of enzymes. Exposure to lead and mercury can cause the development of autoimmunity, which can result in joint diseases, such as rheumatoid arthritis, kidney diseases, circulatory and nervous system disorders, and the damaging of the fetal brain in humans. Exposure to lead and mercury in children causes reduced intelligence, impaired development, and an increased risk of cardiovascular disease. Cadmium is known to be carcinogenic and mutagenic, and can disrupt the endocrine system, damage fragile bones and lungs, and affect the regulation of calcium in biological systems. Chromium causes hair loss, headaches, diarrhea, nausea, and vomiting in humans.

Heavy metal contaminated soils limit plant habitats due to toxicity, resulting in ecological, evolutionary, and nutritional problems, as well as severe selection pressures ^[9][10][9,10]. The toxicity of heavy metals in plants varies, depending on the plant species, specific metal involved, concentration of metal, chemical form of metal, and soil composition and pH [11]. There can be a build-up of heavy metals in plant tissues that affects or inhibits nutrient uptake, homoeostasis, growth, and development. They disrupt metabolic functions, such as physiological and biochemical processes, biochemical lesions, cell organelles destruction, chlorosis, delayed germination, induced genotoxicity, inhibition of photosynthesis and respiration, loss of enzyme activities, oxidative stress, premature leaf fall, reduced biomass, reduced crop yield, senescence, stunted growth, wilting and can even cause the death of plants.

Heavy metal toxicity affects microbial population size, diversity, and activity, as well as their genetic structure. It affects the morphology, metabolism, and growth of microorganisms by altering the nucleic acid structure, disrupting the cell membranes, causing functional disturbance, inhibiting enzyme activity and oxidative phosphorylation, and causing lipid peroxidation, osmotic balance alteration, and protein denaturation^[6][12] [6,12].

3. Bioremediation of Heavy Metals by Microorganisms

Several techniques have been used for the removal and/or recovery of heavy metals from polluted environments. Some established conventional procedures for heavy metal removal and/or recovery from solution, include adsorption processes, chemical oxidation or reduction reactions, chemical precipitation, electrochemical techniques, evaporative recovery, ion exchange, reverse osmosis, and sludge filtration [13]. However, these techniques are expensive, sometimes impracticable, and are not specific for metal-binding properties. Furthermore, the generation of toxic waste, the high reagent requirement, and the unpredictable nature of metal ion removal, highlights some of the disadvantages of these methods. The majority of these methods are ineffective when metal concentrations in solution are less than 100 mg/L^[14] [14]. Separation by physical and chemical techniques is also challenging due to the high solubility of most heavy metal salts in solution.

Bioremediation is an innovative technique for the removal and recovery of heavy metal ions from polluted areas, and involves using living organisms to reduce and/or recover heavy metal pollutants into less hazardous forms, using the activities of algae, bacteria, fungi, or plants. It has been employed for the removal of heavy metals from contaminated wastewaters and soils. This method is an appealing alternative to physical and chemical techniques, and the use of microorganisms play a significant role in heavy metal remediation. Similarly, the use of microorganisms to remediate polluted environments is sustainable and helps to restore the natural state of the polluted environment with long-term environmental benefits and cost effectiveness^[1] [1]. These organisms help to detoxify hazardous components in the environment. The process can function naturally or can be improved through the addition of electron acceptors, nutrients, or other factors.

Detoxification can occur through the valence transformation mechanism. This is particularly applicable in the case of metals whose different valence states vary in toxicity. In mercury-resistant bacteria, organomercurial lyase converts methyl mercury to Hg(II), which is one hundred-fold less toxic than methyl mercury^[15] [15]. The reduction of Cr(VI) to Cr(III) is widely studied, with Cr(III) having less mobility and toxicity. Other detoxification mechanisms of heavy metals are accomplished through metal binding, vacuole compartmentalization, and volatilization. Metal binding involves chelators, such as metallothein, glutathione-derived-peptides called phytochelatin, and metal binding peptides. These chelators bind to heavy metals and facilitate microbial absorption and the transportation of metal ions. Volatilization mechanisms involve turning metal ions into a volatile state. This is only possible with Se and Hg, which have volatile states. Mercury-resistant bacteria utilizes the MerA enzyme to reduce Hg(II) to the volatile form Hg(0)^[15] [15]. The reduction of Se(V) to elemental Se(0) has been employed to remediate contaminated waters and soils. The metabolic processes of these organisms help to transform pollutants in the environment^[13] [13].

Biosorption, bioaccumulation, biotransformation, and biomineralization are the techniques employed by microorganisms for their continued existence in metal polluted environment. These strategies have been exploited for remediation procedures^[16][17] [16,17]. Living organisms or dead biological materials can carry out heavy metal removal. Large-scale feasibility applications of biosorptive processes have shown that dead biomass is more applicable than the bioaccumulation approach, which involves the use of living organisms and thus requires nutrient supply and a complicated bioreactor system. In addition, the toxicity of pollutants, as well as other unfavorable environmental conditions, can contribute to the inability to maintain a healthy microbial population. However, many characteristic attributes of living microorganisms have not been exploited in large-scale applications^[18] [18]. The choice organism must develop resistance towards metal ions as it is exposed to the heavy metal pollutant to achieve the goal of remediation. The organism of choice may be native to the polluted environment or isolated from another environment and brought to the contaminated site^[19] [19].

Advances in the understanding of metabolic pathways of microorganisms are responsible for metal sequestration, improving microbial survival rates, and their stability. This has led to the manipulation of metal adsorption^[20] [20]. Adsorption is the physical adherence of ions and molecules onto the surface of another molecule. The material accumulated at the interface is the adsorbate and the solid surface is the adsorbent. If adsorption occurs and results in the formation of a stable molecular phase at the interface, this can be described as a surface complex. Most solids, including microorganisms, possess functional groups like –SH, –OH, and –COOH on their surfaces, that helps in the adsorption of metals^[21] [21]. It has been reported that a microbial cell develops resistance to heavy metals through the excretion of metal chelating substances, or through a problem in a particular transport system, which results in a reduced cell accumulation of the metal ion. Another resistance mechanism includes the binding of a metal ion to intracellular molecules, such as metallothionein, vacuole, or mitochondria, which results in changes in the distribution of metal ion^[13] [13]. Microorganisms interact with metal ions through cell wall associated metals, intracellular accumulation, metalsiderophore, extracellular polymeric reactions with transformation, extracellular mobilization or immobilization of metal ions, and volatilization of metals^[13] [13].

Various factors influence the microbial remediation of metals. They include the bioavailability of the metal to the microbe, concentration of pollutants, electron acceptors, moisture content, nutrients, osmotic pressure, oxygen, pH, redox potential, soil structure, temperature, and water activity. The bioavailability of each metal in soil is influenced by factors such as the buffering capacity, cation exchange capacity, clay minerals content, metal oxide, and organic matter^[9][22][23] [9, 22,23]. In general, remediation of heavy metal is achieved through the removal of the metal ion from substratum to reduce the risk posed by exposure to such heavy metals.

4. Mechanisms of Heavy Metal Uptake by Microorganisms

The cellular structure of a microorganism can trap heavy metal ions and subsequently sorb them onto the binding sites of the cell wall^[24] [24]. This process is called biosorption or passive uptake, and is independent of the metabolic cycle. The amount of metal sorbed depends on the kinetic equilibrium and composition of the metal at the cellular surface. The mechanism involves several processes, including electrostatic interaction, ion exchange, precipitation, the redox process, and surface complexation^[25] [25]. The process is fast and can reach equilibrium within a few minutes. Biosorption can be carried out by fragments of cells and tissues, or by dead biomass or living cells as passive uptake via surface complexation onto the cell wall and other outer layers [26]. The other method is a process in which the heavy metal ions pass across the cell membrane into the cytoplasm, through the cell metabolic cycle. This is referred to as bioaccumulation or active uptake. Bioaccumulation is a process of a living cell that is dependent on a variety of physical, chemical, and biological mechanisms. These factors include intracellular and extracellular processes, where biosorption plays a limited and ill-defined role^[26] [26]. The organism that will accumulate heavy metals should have a tolerance to one or more metals at higher concentrations, and must exhibit enhanced transformational abilities, changing toxic chemicals to harmless forms that allows the organism to lessen the toxic effect of the metal, and at the same time, keep the metal contained^[27] [27].

Metal uptake mechanisms by various biosorbents depend on the cellular surface of the microbes, as well as the exchange of metal ions and complex formations with the metal ions on the reactive chemical sites of the cell surface. These have been extensively studied with respect to various biosorption isotherms, derived from sorption experiments and the effect of various factors, such as pH, biomass pretreatment, and the biomass of the organisms. Precipitation of the excess metal ions, through nucleation reactions, then occurs at the cell surface. All microorganisms have a negative charge on their cell surface due to the presence of anionic structures, which enable them to bind to metal cations. The negatively charged groups that are involved in metal adsorption are the alcohol, amine, carboxyl, ester, hydroxyl, sulfhydryl, phosphoryl, sulfonate, thioether, and thiol groups^[20] [20].

An analysis of the cell wall components, which vary among the different microorganisms, helps in assessing metal uptake by different microorganisms. The peptidoglycan layer in Gram-positive bacteria, which contains alanine, glutamic acid, meso-di-aminopimelic acid, polymer of glycerol and teichoic acid, and that of the Gram-negative bacteria, which contains enzymes, glycoproteins, lipopolysaccharides, lipoproteins, and phospholipids, are the active sites involved in metal binding processes^[26][28][29] [26,28,29]. Metals and metalloids are attached to these ligands on cell surfaces, which displace essential metals from their normal binding sites. Once the metal and metalloid are bound, microbial cells can transform them from one oxidation state to another, thus reducing their toxicity^[30] [30]. Gavrilescu^[20] [20] reported that the cell walls of bacteria are polyelectrolyte, which interacts with metal ions to maintain electro-neutrality by mechanisms of covalent bonding, extracellular precipitations, redox interactions, and van der Waals forces.

The rigid cell wall of fungi is made up of chitin, inorganic ions, lipids, nitrogen-containing polysaccharide, polyphosphates, and proteins. They can tolerate and detoxify metal ions by active uptake, extracellular and intracellular precipitation, and valence transformation, with many absorbing heavy metals into their mycelium and spores. The surface of their cell wall acts as a ligand for binding metal ions, resulting in the removal of metals^[29] [29]. The first barrier includes excreted substances like organic acids or/and proteins with an ability to immobilize heavy metals. The second barrier includes the (unspecific) binding of heavy metals by the cell wall and melanins located in the cell wall. Toxic heavy metals that could not be detained outside the cell must be detoxified inside the cell^[31] [31].

The cell wall of all classes of algae is composed of cellulose with sulfonated polysaccharides present in the cell wall of brown and red algae. Other binding sites in algae are polysaccharides such as alginic acid, glycan, mannan, proteins, and xylans. The cell wall of cyanobacteria is composed of peptidoglycan, and some species produce sheaths and extracellular polymeric substances, which are used for sorption. Characteristics of the biomass, chemical and physical properties of the metal of interest, and pH of the solution, influence the sorption capacity of algae^[28] [28].

Non-essential metal uptake usually consists of transporters, which are committed to the acquisition of vital organic and inorganic ions. These transporters assist either in the co-transport of these metals in complexes with low-molecular-mass ligands, or in the direct uptake of non-essential metals^[32] [32]. Microorganisms can also secrete many kinds of metal-binding metabolites, produce extracellular polymeric substances, which are made up of polysaccharide, capsules, slimes and sheaths, and biofilms, depending on the make-up of the polysaccharide and associated components. Biofilms bind substantial quantities of heavy metals under pristine conditions and serve as a medium for the precipitation of insoluble mineral phases^[26] [26].